【作者】 秦建；

【导师】 王远亮；

【作者基本信息】 重庆大学 ， 细胞生物学， 2007， 硕士

【摘要】 肿瘤是危害人类健康最严重的疾病之一,其侵袭转移过程存在着复杂的生物流变学问题。目前,肿瘤的治疗方法主要有化学治疗、放射治疗、生物治疗、电穿孔治疗及外科手术治疗等,其中电穿孔疗法是近年来发展起来的一种杀伤肿瘤组织/细胞的新技术,并逐渐成为癌症综合治疗的有效手段之一,但人们对于脉冲电场治疗肿瘤的机理至今尚缺乏全面的认识。本项目将生物流变学和电工理论新技术有机结合,采用陡脉冲电场在一定条件下作用肝癌细胞,首先考查陡脉冲电场作用后肝癌细胞生长、增殖、凋亡相关基因的表达变化及其相关规律,然后用微管吸吮技术在单个细胞水平上研究陡脉冲电场作用后肝癌细胞粘弹特性、与内皮细胞粘附特性的变化情况,从生物流变学的角度探索陡脉冲电场杀伤肿瘤细胞的机理。主要研究工作和结果如下:1.陡脉冲电场实验装置及刺激参数能量可控陡脉冲实验装置由重庆大学高电压及电工理论新技术教育部重点实验室自行研制,该仪器主要由直流高压产生电路、陡脉冲的形成与整形输出电路和计算机采集测量系统三部分组成。根据陡脉冲电场杀伤肿瘤细胞/组织的前期研究基础,本文选择固定脉冲峰值(200 V)、频率(100 Hz)及刺激时间(10min) 3个参数,改变电容值(取0.01μF和0.1μF高低两种刺激剂量)观察陡脉冲电场对肝癌细胞增殖和生物流变特性的影响。2.陡脉冲电场对肝癌细胞生长和增殖的影响采用MTT法分析了陡脉冲电场作用后肝癌细胞增殖的变化,流式细胞技术分析了细胞周期分布的变化。结果表明:陡脉冲电场对肝癌细胞生长和增殖有明显的抑制作用;高剂量的陡脉冲电场抑制程度更为明显。肝癌细胞经陡脉冲电场作用后,细胞周期分布发生改变,细胞G0/G1期增加,S期和G2/M期减少。提示陡脉冲电场刺激能阻止肝癌细胞进入S期。3.陡脉冲电场对肝癌细胞凋亡相关基因表达的影响采用流式细胞技术考察了陡脉冲电场作用后肝癌细胞bcl-2和p53基因表达的变化,结果表明:陡脉冲电场能下调肝癌细胞bcl-2基因的表达,但p53基因的表达不受陡脉冲电场的影响,提示陡脉冲电场诱导肝癌细胞凋亡,可能是通过降低bcl-2的表达实现的。4.陡脉冲电场对肝癌细胞粘弹性的影响与对照组比较,低剂量陡脉冲电场处理组的弹性系数K1没有明显变化,但高剂量处理组的弹性系数K1明显降低;低剂量处理组的弹性系数K2没有明显变化,但高剂量处理组的弹性系数K2明显升高;粘性系数μ在陡脉冲电场处理后都有所增高,但高剂量处理组比低剂量处理组增高的程度更大。陡脉冲电场处理后,各组细胞初始变形程度没有差别;但与对照组比较,高剂量处理组细胞的最大变形程度有所增高;陡脉冲电场作用后细胞蠕变时间增长,高剂量组增加的程度更明显。5.陡脉冲电场对肝癌细胞与内皮粘附力的影响与对照组比较,低剂量刺激组肝癌细胞与内皮细胞的粘附力明显降低,而高剂量刺激组的粘附力没有明显变化。更多还原

【Abstract】 Malignant tumor is one of the most severe diseases which does harm to human’s health. At present, the main treatments for tumor include chemical therapy, radiation therapy, biologic therapy and electroporotion therapy. Electroporotion therapy is a new developed technique to kill malignant tissues or cells and has become one of the efficient methods of tumor comprehensive treatment. However there is lack of all-round understanding about the mechanisms of electroporotion therapy to tumor.In this thesis, we combined biorheology and electrical technique, and investigated the growth, proliferation and apoptosis related gene expression of hepatoma cells treated by EPEF. We also studied the change of viscoelasticity、adhesive force of hepatoma cells to human umbilical vein endothelial cells(HUVECs) using of micropipette aspiration technique. The main works and results in this paper are as follows:1. SPEF equipment and experimental parametersSPEF equipment was self-assembled by Key Laboratory of High Voltage & Electrical New Technique of Ministry of Education of China, Chongqing University. It consists of circuit of direct current, producing and output of SPEF and collecting and processing system controlled by computer. In our experiments, we fixed voltage(200V), frequency (100Hz), treanment time (10min) and changed the capacitance values (0.01μF and 0.1μF)to investigate the effects of SPEF on biorheological properties.2.Effects of SPEF on proliferating dynamics of hepatoma cellsWith MTT assay, the growth and proliferation of hepatoma cells treated by SPEF were evaluted. Results showed that SPEF could inhibit the growth and proliferation of hepatoma cells and high dosage treatment showed a stronger inhibition than that of low dasage treatment. It also showed that G0/G1 phase increase but S phase and G2/M phase decrease after treated by SPEF, which suggested that SPEF could change the distribution of cell cycle and block hepatoma cells entering S phase.3. Effects of SPEF on expression of gene involved apoptosis in hepatoma cellsAfter exposure to SPEF, Expression of bcl-2 and p53 gene in hepatoma cells was measured by FCM and FITC marked monoclonal antibody quantificationally. It found that SPEF could down-regulate the expression of bcl-2 gene, but expression of p53 gene don’t have any change. It indicated that the apoptosis of hepatoma cells induced by SPEF maybe performed through down-regulate the expression of bcl-2 gene. 4. Effects of SPEF on viscoelastical properties of hepatoma cellsCompared with control, both K1 and K2 values of hepatoma cells treated by low dosage SPEF have no change, while K1 of hepatoma cells treated by high dosage SPEF showed an obvious decrease and K2 significantly increase. Viscous coefficientμshowed an increase trend in all treatment groups after exposure to SPEF. As for the eigenvalues of viscoelasticity of hepatoma cells, it showed that the primary deformation have no defference but the maximum deformation of high dasage treatment hepatoma cells increase markedly when compared with control cells. It also showed a smaller rigidity and longer time of creeping deformation than that of control cells.5. Effects of SPEF on adhesive force of hepatoma cells to HUVECsAdhesive force of low dasage treatment cells to HUVECs present a higher value when compared with control cells. However, adhesive force of high dasage treatment cells to HUVECs reveals no significant difference compared with control cells.更多还原